Cell culture medium is used to keep cells alive outside of an organism, usually in a laboratory (terminology is in vitro; Latin for “in glass”). It is composed of a sterile nutrient system; usually a complex mixture of organic and inorganic materials. Protein growth factors are usually added to promote cell growth or other specific behaviors. Supplements are added to protect the cells from microorganisms or stabilize the environment.
Cell culture media is generally stored in clear plastic or glass bottles with screw tops or in some cases, bags. Labeling commonly consists of a stick-on paper or plastic based label that covers a limited area of the container (generally less than 25 percent of the container's surface area).
Media is added to cells in liquid form. As living cells metabolize components of the medium critical components are depleted and waste products are produced, creating the need for frequent media replacement.
As a liquid, media should be stored at refrigeration temperatures and be shielded from light. Low temperatures decelerate the normal rates of protein denaturation that can deplete critical media components. Protection from visible light is necessary because light exposure leads to photoreactions involving riboflavin, thiols, metal ions and other components that can degrade the component and produce reactive oxygen species (ROS) that damage cells (see Grzelak et al., “Light-dependent generation of reactive oxygen species in cell culture media”, Free Radic. Biol. Med. 12:1418-1425; 2001) through various mechanisms, leading to DNA modifications or mutations. Room fluorescent light has been found to generate phototoxic products including hydrogen peroxide (see Stoien et al., “Effect of near-ultraviolet and visible light on mammalian cells in culture II. Formation of toxic photoproducts in tissue culture medium by blacklight”, Proc. Natl. Acad. Sci. USA 71:3961-3955; 1974; Wang et al., “Effect of room fluorescent light on the deterioration of tissue culture medium”, In Vitro. 12:19-22; 1976; and Wang et. al., “Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue-culture medium irradiated with “daylight” fluorescent light”, In Vitro. 14:715-722; 1978). Common media components such as tryptophan, tyrosine, pyridoxine and folic acid can enhance these reactions (see Grzelak et al., supra). The presence of serum in medium has historically minimized the problems caused by light-produced ROS. However, in the defined, serum-free or low-serum media commonly used in mammalian cell culture today, ROS-generated cytotoxicity can become more prevalent.
Media is applied to mammalian cells at a temperature of 37° C. to provide an ideal environment for cell metabolism to take place. Applying media at a temperature that is too low can shock the cells leading to inhibited metabolism or reduced viability. To avoid these problems cell culture media is usually warmed before it is applied to cells. Generally, cell culture medium is warmed in a water bath by immersing a container containing the medium into water in a “water bath” that has been pre-warmed to a pre-determined temperature. If small volumes of medium are needed, the technicians transfer a portion of the medium from the original container into a second sterile container (using sterile technique) or by warming the entire original bottle in the water bath. Currently the common practice is for cell culture technicians to estimate when the media inside the container has reached the proper temperature. This is usually done by timing or by touching the outside of the container to determine if it “feels” to be the proper temperature. If the technician incorrectly estimates the medium temperature the cells may be inhibited metabolically, have reduced viability or in extreme cases perish due to the shock caused by large variations in medium temperature over a short period of time.
Contamination may occur during the media-warming process because the media container is directly exposed to non-sterile water in the water bath. Bacteria, mold, yeast and other microorganisms present in the non-sterile water bath water remain on the outside of the media container and may be carried on the outside of the media container into a sterile environment, such as a biological safety cabinet, where the warmed media is transferred into cell cultures. Technicians usually minimize this hazard by washing the outside of the media bottle with sterilizing solutions such as 70% isopropyl alcohol.
What is needed is a way of storing and manipulating cell culture medium to avoid the problems outlined above.